While it is clear that HIV-1 assembly occurs predominantly on the plasma membrane (PM), the itinerary that the Gag precursor follows to reach its destination remains ill defined. Likewise, the host cell machinery that promotes Gag trafficking to the PM is incompletely understood. We and others have demonstrated that the matrix (MA) domain of Gag regulates targeting to the site of virus assembly, and we discovered that the phosphoinositide PI(4,5)P2 and the ADP ribosylation factors (Arfs) serve important functions in directing Pr55Gag to the PM. We are actively engaged in studies to elucidate further the cellular machinery involved in HIV-1 Gag trafficking. This effort combines virology, biochemistry, cell biology, and imaging techniques and is also being extended to the nonprimate lentiviruses equine infectious anemia virus (EIAV) and feline immunodeficiency virus (FIV). Proteins of particular interest in this area include the Arf proteins, SNAREs, F-BAR domain proteins, clathrin, and FLJ90680. In studies related to Gag trafficking, we are also investigating the host cell machinery required for Gag localization to, and virus transfer across, the virological synapse. The viral envelope (Env) glycoproteins are incorporated into virions during the assembly process. We have shown that point mutations in the MA domain of Gag block Env incorporation, and demonstrated that the long gp41 cytoplasmic tail plays a cell-type-dependent role in Env incorporation. Despite the evidence for a direct interaction between MA and the gp41 cytoplasmic tail, the molecular mechanism by which Env is incorporated and the basis for cell type dependence of cytoplasmic tail function remain to be defined. We will address the hypothesis that host cell factors are likely to play an important role in Env incorporation into virions. We have developed a bimolecular fluorescence complementation (BiFC) assay for Gag-Env interaction that will be useful in evaluating this hypothesis. We have also demonstrated that MA trimerization plays a critical role in Env incorporation and have identified residues that are required for MA trimer formation. [Corresponds to Freed Project 1 in the October 2011 site visit report of the HIV Drug Resistance Program]